Apolipoprotein B is both integrated into and translocated across the endoplasmic reticulum membrane. Evidence for two functionally distinct pools

Apolipoprotein B (apoB), a protein containing several hydrophobic beta-sheet structures, is essential for the assembly of triglyceride-rich lipoproteins. Previously, we found that only a fraction of de novo synthesized apoB is secreted; the remainder is retained in the endoplasmic reticulum where it is degraded. To understand the basis for these observations, translocation, the first step in the secretory pathway, was examined. Translocation of apoB was determined by its sensitivity to degradation by the exogenous protease, trypsin. In rough microsomes, about half of the apoB was degraded by trypsin. In contrast, in Golgi fractions little (if any) apoB was accessible to trypsin. Essentially all of the apoB that was degraded was membrane bound. Monoclonal IgGs against either the N-terminal or C-terminal halves of apoB were bound to magnetic beads and used to immunoisolate microsomes. In contrast to the specific ability of the IgGs against apoB to isolate microsomes, little or no microsomes were isolated using nonimmune IgG and IgG against albumin. Since microsomes remained intact and oriented right-side out as demonstrated by the inability of trypsin both to degrade albumin and to affect the capacity of the intralumenal enzyme glucose-6-phosphatase to dephosphorylate mannose 6-phosphate, the data suggest that a pool of apoB is exposed on the cytoplasmic surface of the endoplasmic reticulum membrane. To determine if the trypsin-accessible pool of apoB is a transient form, pulse-chase experiments were performed. The results show that the percent of apoB that was trypsin accessible increased during the first 20 min of the chase, suggesting that during this time the trypsin-accessible pool of apoB is not translocated (it does not become trypsin insensitive). Thus, in two in vivo models (cultured cells and rat liver) translocation of apoB is not quantitative. We propose that apoB translocation across the endoplasmic reticulum determines its entry into two functionally distinct pools. The intralumenal trypsin-insensitive pool participates in the assembly of very low density lipoprotein; the trypsin-accessible nontranslocated cytoplasmic pool is shunted into a degradative pathway. Regulated translocation of apoB may provide a unique mechanism with which to determine the rate of very low density lipoprotein assembly/secretion.     

Intrahepatic assembly of very low density lipoproteins: immunologic characterization of apolipoprotein B in lipoproteins and hepatic membrane fractions and its intracellular distribution

Monoclonal antibodies, prepared against rat apoB, were used to examine apoB structure in serum lipoproteins and characterize the forms and localization of apoB in liver membrane fractions and cultured hepatocytes. Of the several antibodies obtained, four, having separate epitopes, were characterized. Western blot analysis showed that three (DB11, F4, and LB14) antibodies recognized both apoBL and apoBS. One antibody (HB41) recognized only apoBL. This antibody showed unusual properties. Competition ELISA assays showed that the epitope recognized by HB41 was more effectively expressed on low density lipoproteins (LDL) compared to very low density lipoproteins (VLDL). In addition, treatment of lipoproteins with detergents and sulfhydryl reducing agents also increased the expression of the HB41 epitope. Since HB41 has been found to inhibit LDL binding to hepatocyte receptors, these data indicate that the HB41 epitope is located on the carboxy-terminal side of the apoBS junction (probably within the LDL receptor binding domain). Western blotting hepatic microsomal subfractions showed that in the rough and smooth microsomes, HB41 recognized only apoBL, while in the Golgi it recognized both apoBL and a protein having a molecular weight slightly smaller. In contrast, Western blotting with a polyclonal antibody known to recognize both apoBL and apoBS showed that, in rough and smooth microsomes, proteins in addition to apoBL and apoBS having molecular weights between 120,000 and 30,000 were recognized. These proteins, likely to be proteolytic fragments of apoB, were barely detectable in the Golgi. Additional biosynthetic studies show that the [35S]methionine-labeled proteins smaller than apoB were immunoprecipitated from the rough microsome subfraction. Pulse-chase experiments show that these are produced with the same kinetics as full-size apoBL and apoBS, indicating that they are not incomplete nascent chains. Finally, immunofluorescence microscopy was used to determine the localization of monoclonal epitopes. ApoB monoclonal antibodies that recognized exclusively apoBL (HB41) and apoBL and apoBS (DB11) produced an immunofluorescence pattern characteristic of the endoplasmic reticulum, but not the Golgi. These data suggest that, in cultured rat hepatocytes, the majority of both molecular weight forms of apoB are localized in the endoplasmic reticulum, the initial site of VLDL assembly. The additional finding that proteolytic fragments of apoB are enriched in the microsomal fraction suggests that if the proteolysis occurs during subcellular fractionation, immature apoB is susceptible to proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural immunolocalization of apolipoprotein B within human jejunal absorptive cells

Apolipoprotein B (apoB) was localized by electron microscopy within absorptive cells of human jejunal biopsy specimens taken fasting and after micellar fat infusion. Nakane's double antibody immunoperoxidase technique was used to label apoB near open cut surfaces of 60-Micrometers fixed tissue slices sectioned by a Ralph knife in a Vibratome. In fasting tissue, apoB label was found within structurally intact peri-mitochondrial rough endoplasmic reticulum (RER) and within Golgi cisternae of absorptive cells covering the tips of jejunal villi. After fat infusion, apoB label was found adjacent to very low density lipoproteins (VLDL) and chylomicrons within apical smooth endoplasmic reticulum (SER). Less label was seen within RER than in fasting absorptive cells, and RER-SER connections containing apoB label were occasionally seen. Expanded Golgi vesicles and cisternae contained VLDL, chylomicrons, and apoB label. Vesicles containing chylomicrons and apoB label were occasionally visualized bordering the lateral plasma membrane in a configuration suggesting exocytosis. Specific apoB label was regularly seen within intercellular spaces and capillaries, but the in vivo significance of this Localization was problematical. These observations suggest that apoB is synthesized in RER, transfers to SER where it is incorporated into new VLDL and chylomicrons, and moves to Golgi cisternae and vesicles to be prepared for exocytosis through the plasma membrane.     

The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.     

Dietary fish oils inhibit early events in the assembly of very low density lipoproteins and target apoB for degradation within the rough endoplasmic reticulum of hamster hepatocytes

Dietary fish oils inhibited secretion and stimulated intracellular degradation of apolipoprotein (apo)B in hamster hepatocytes, while dietary sunflower oils stimulated secretion and had no effect on degradation of apoB. To investigate the intracellular site at which fish oils act, we have made use of our previous observations that inhibition of degradation by N-acetyl-leucyl-leucyl-norleucinal (ALLN) results in accumulation of apoB in the trans -Golgi membrane and does not stimulate secretion, while inhibition of degradation by o-phenanthroline results in accumulation of apoB in the rough endoplasmic reticulum membrane and stimulates secretion. Thus, ALLN protects apoB which has been diverted from secretion and o -phenanthroline protects apoB which is targetted for secretion. Addition of o -phenantholine to the incubation medium of hepatocytes from fish oil-fed hamsters inhibited degradation of apoB and stimulated its secretion in particles of the density of VLDL, while addition of ALLN had no effect.These observations suggest that dietary fish oils reversibly inhibit early steps in the assembly of very low density lipoprotein precursors and target apoB for degradation in the rough endoplasmic reticulum.     

The assembly and secretion of apoB 100 containing lipoproteins in Hep G2 cells. Evidence for different sites for protein synthesis and lipoprotein assembly

Pulse-chase studies combined with subcellular fractionation indicated that LpB 100 (i.e. the apoprotein B (apoB) 100 containing lipoproteins) was released to the lumen of the secretory pathway in a subcellular fraction enriched in smooth vesicles, and referred to as SMF (the smooth membrane fraction). The migration of SMF during gradient ultracentrifugation as well as kinetic studies indicated that the fraction was derived from a pre-Golgi compartment, probably the smooth endoplasmic reticulum (ER). Only small amounts of LpB 100 could be detected during these pulse-chase experiments in the subcellular fractions derived from the rough endoplasmatic reticulum (RER). SMF contained the major amount of the diacylglycerol acyltransferase activity present in the ER, while the major amount of membrane bound apoB 100 was present in the RER. Pulse-chase studies of the intracellular transfer of apoB 100 demonstrated the formation of a large membrane-bound preassembly pool in the ER, while no significant amount of apoB 100 radioactivity was present in the membrane of the Golgi apparatus. The maximal radioactivity of LpB 100, recovered from the ER or the Golgi lumen, was small compared with the radioactivity recovered from the ER membrane, indicating that the assembled LpB 100 rapidly leaves the cells. This in turn indicates that the rate-limiting step in the secretion of apoB 100 was the transfer of the protein from the ER membrane to the LpB 100 in the lumen. A portion of the intracellular pool of apoB 100 was not secreted but underwent posttranslational degradation.     

Transmembrane lipid transfer is crucial for providing neutral lipids during very low density lipoprotein assembly in endoplasmic reticulum

Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [14C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [14C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.     

Localization of proteoglycan core protein in subcellular fractions isolated from rat chondrosarcoma chondrocytes

Chondrocytes from the Swarm rat chondrosarcoma were pulse-labeled with [3H]serine for 30 min and chased, in the presence of cycloheximide, for times up to 300 min. The movement of newly synthesized core protein precursor of the proteoglycan through elements of the endoplasmic reticulum and Golgi complex was examined. Rough and smooth microsome fractions were obtained by centrifuging postmitochondrial supernatants from cell homogenates on discontinuous sucrose gradients. The core protein precursor was identified in subcellular fractions by (a) immunoprecipitation with an antiserum directed against the hyaluronate binding region of the core protein and the link protein and (b) its size on polyacrylamide gels. Labeled core protein precursor decreased from the microsomes with a t1/2 of 60 +/- 8 min, nearly the same as for the appearance of label in completed proteoglycan monomer (t1/2 = 58 +/- 13 min), consistent with a precursor-product relationship. After correcting for incomplete recovery of the core protein precursor in the microsomal fractions and for cross-contamination of the smooth microsomes by elements of rough endoplasmic reticulum, the redistribution of core protein precursor and completed proteoglycan in the intracellular compartments and of labeled extracellular proteoglycan were fit to a three-compartment model. A t1/2 of 98 +/- 7 min for the loss of core protein precursor from the rough microsomes and a t1/2 = 10 +/- 4 min for the completed proteoglycan in the intracellular compartment (Golgi and secretory vesicles) was obtained. The data indicate that at least 70% of the intracellular transit time for the core protein precursor is spent in the rough endoplasmic reticulum. The addition of glycosaminoglycan chains followed by secretion from the cell occurs relatively rapidly, occupying less than 30% of the total intracellular dwell time.     

Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells

Very low-density lipoprotein (VLDL) particles are formed in the endoplasmic reticulum (ER) through the association of lipids with apolipoprotein B (apoB). Microsomal triglyceride transfer protein (MTP), which transfers lipid molecules to nascent apoB, is essential for VLDL formation in ER. However, little is known of the distribution and interaction of MTP with apoB within ER. In this study, distribution patterns of apoB and MTP large subunit (lMTP) within ER were examined. Microsomes prepared from HuH-7 cells, a human hepatoma cell line, were further fractionated into rough ER (RER)-enriched subfractions (ER-I fraction) and smooth ER (SER)-enriched subfractions (ER-II fraction) by iodixanol density-gradient ultracentrifugation. ApoB was evenly distributed in the ER-I and the ER-II fractions, while 1.5 times more lMTP molecules were present in the ER-I fraction than in the ER-II fraction. lMTP and apoB were coprecipitated both in the ER-I and in the ER-II fractions by immunoprecipitation whenever anti-apoB or an anti-lMTP antibodies were used. ApoB-containing lipoprotein particles showed a lower density in the ER-II fraction than those in the ER-I fraction. From these results, it is suggested that MTP can function in both rough and smooth regions of ER in human hepatoma cells.     

The biosynthesis of rat transferrin. Evidence for rapid glycosylation, disulfide bond formation, and tertiary folding

The transit time of newly synthesized transferrin in the liver is markedly longer than that of albumin. We sought to learn the basis of this difference by the use of labeled leucine and mannose in vivo and by isolation of newly formed transferrin from rough microsomes of rat liver. Albumin and alpha 1-antitrypsin, a second glycoprotein, were also studied for comparison. Minimal hepatic transit times were 17, 23, and 31 min for albumin, alpha 1-antitrypsin, and transferrin, respectively. The delay in the case of transferrin was found to occur chiefly in the rough endoplasmic reticulum and to be paralleled by an increase in the amount of transferrin relative to albumin in that organelle. Initial glycosylation of transferrin was as rapid as that of alpha 1-antitrypsin, and essentially all of the transferrin in the rough endoplasmic reticulum contained glycans which bound to concanavalin A and were removed by endoglycosidase H. Only 3% of the transferrin isolated from the rough microsomes came from the plasma by endocytosis or adsorption. Rapidity of disulfide bond formation in rough microsomes was evident from the presence of only 1.3 cysteine thiols/molecule of rough microsomal transferrin (total of 19 cystines) and the absence of mixed disulfides. Peptide patterns upon mild proteolysis were consistent with a native configuration of disulfide bond pairing. The ability of rough microsomal transferrin to bind and deliver iron through interaction with transferrin receptors on reticulocytes suggests that considerable tertiary structure is present. Thus, initial glycosylation, disulfide bridging, and tertiary folding are all rapid processes. The cause for the slow release of transferrin from the rough endoplasmic reticulum may lie with a rate-limiting transfer mechanism.     

Interferon suppresses glutamine synthetase induction in chick embryonic neural retina.

Two different proteins precipitable with antiserum to albumin exist in liver. One is albumin, the other is precursor albumin. Liver cells in suspension contain mainly precursor, but secrete only albumin. In subcellular fractions isolated from liver homogenate, 95.3% of anti-albumin precipitable protein in the rough endoplasmic reticulum, 51.4% in the smooth endoplasmic reticulum, 33.5% in the Golgi apparatus and 0% in the supernatant fraction was precursor albumin. The results suggest that albumin precursor is synthesized in the rough endoplasmic reticulum and converted into albumin in the smooth endoplasmic reticulum and the Golgi apparatus.     

SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY : II. Electrophoretic and Immunological Characterization of Microsomal Subfractions

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [³H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [³H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [³H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.     

The isolation of microsomal subfractions of mouse plasmacytoma cells: The effect of salt concentration during nitrogen cavitation

Following disruption of MPC-11 cells by nitrogen cavitation the microsomes have been fractionated by centrifugation on discontinuous sucrose gradients. When the homogenization buffer contained 25 mm KCl three fractions were observed: smooth microsomes, light rough microsomes, and heavy rough microsomes. When it contained 100 mm KCl, however, only smooth and light microsomal fractions were found. Under the latter conditions the heavy rough microsomal vesicles were apparently not released as separate organelles but instead sedimented together with the endoplasmic reticulum which remains attached to the nuclei.     

Regulation of apoB secretion from HepG2 cells: evidence for a critical role for cholesteryl ester synthesis in the response to a fatty acid challenge

Secretion of hepatic apoB lipoproteins removes excess triglyceride from the liver. However, the mechanism by which synthesis of apoB, which occurs on the rough endoplasmic reticulum, is coordinated with synthesis of triglyceride, which takes place in the smooth endoplasmic reticulum, is not known. To examine this question, we have manipulated intracellular synthesis of triglyceride and cholesteryl ester in HepG2 cells and determined the impact of these maneuvers on apoB secretion. Since cholesteryl ester is the only major lipid class synthesized in the rough endoplasmic reticulum, our hypothesis was that, in response to a fatty acid challenge, synthesis of cholesteryl ester rather than synthesis of triglyceride would be the immediate trigger to apoB secretion. Oleate complexed to bovine serum albumin caused intracellular triglyceride synthesis to increase 6-fold and cholesteryl ester synthesis to increase almost 3-fold, while apoB secretion into the medium increased by 2.5-fold (P less than 0.0125) at all time points between 4 and 24 h. Addition of acylation stimulating protein to the medium further stimulated both triglyceride and cholesteryl ester synthesis (58% and 108%, respectively) above oleate alone and this resulted in a 50% increase in apoB secretion (P less than 0.0025). By contrast, both progesterone and 2-bromooctanoate inhibited triglyceride and cholesteryl ester synthesis and these effects were associated with reduced apoB secretion. Lovastatin inhibited cholesteryl ester synthesis (45%, P less than 0.0025); however, at the doses used, triglyceride formation was unaffected. Under these circumstances, apoB secretion was reduced by 25% (P less than 0.05). Similarly, 58-035 (an inhibitor of acyl CoA:cholesterol acyltransferase) on the one hand reduced cholesteryl ester synthesis markedly (59%, P less than 0.005), but on the other increased triglyceride synthesis though not statistically significantly (65%, P NS), and again this resulted in decreased apoB secretion (25%, P less than 0.005). Control experiments established that changes in low density lipoprotein catabolism did not contribute importantly to the quantity of apoB in the medium. Taken together, the data indicate that, at least in HepG2 cells, there are parallel changes in cholesteryl ester synthesis and apoB secretion and suggest that it is cholesteryl ester synthesis, not triglyceride synthesis, that is the immediate regulator of apoB secretion when these cells are exposed to an increased influx of fatty acids. However, alternative or additional regulatory mechanisms, such as, for example, a role for acylation of apoB, are not excluded by these studies.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : I. Isolation of Membrane Fractions

The subcellular components involved in the synthesis, transport, and discharge of secretory proteins in the guinea pig pancreatic exocrine cell have been isolated from gland homogenates by differential and gradient centrifugation. They include rough and smooth microsomes derived respectively from the rough endoplasmic reticulum and Golgi periphery, a zymogen granule fraction consisting mainly of mature zymogen granules and a smaller population of condensing vacuoles, and a plasmalemmal fraction. Membrane subfractions were obtained from the particulate components by treatment with mild (pH 7.8) alkaline buffers which extract the majority (>95%) of the content of secretory proteins, allowing the membranes to be recovered from the extracting fluid by centrifugation. The purity of the fractions was assessed by electron microscopy and by assaying marker enzymes for cross-contaminants. The rough and smooth microsomes were essentially free of mitochondrial contamination; the smooth microsomes contained <15% rough contaminants. The zymogen granule fraction and its derived membranes were free of rough microsomes and contained <3% contaminant mitochondria. The plasmalemmal fraction was heterogeneous as to origin (deriving from basal, lateral, and apical poles of the cell) and contained varying amounts of adherent fibrillar material arising from the basement membrane and terminal web. The lipid and enzymatic composition of the membrane fractions are described in the following reports.     

Biogenesis of endoplasmic reticulum membrane in rat liver cells. II. Discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 on the cytoplasmic side of the endoplasmic reticulum membrane.

The direction of discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 from bound polyribosomes of rough microsomes was investigated in order to elucidate the mechanism of separation of these membrane proteins from secretory proteins, which are also synthesized by the same class of ribosomes of rough endoplasmic reticulum. The nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 in intact rough microsomes were accessible to externally added 125I-Fab's against these proteins, and were susceptible to trypsin digestion, whereas the nascent peptides of serum albumin were not. The nascent peptides of these two microsomal proteins were released into the cytoplasm by puromycin treatment of intact rough microsomes, while the nascent peptides of serum albumin were retained in the microsomal lumen. These observations suggest that the nascent peptides of microsomal proteins, which are present on the cytoplasmic surface of the endoplasmic reticulum membrane, are exposed on the surface of microsomal vesicles, while those of secretory proteins are enclosed inside the vesicles. Therefore, the topographical separation of microsomal membrane proteins from secretory proteins is accomplished at the step of their synthesis by the bound polyribosomes of rough endoplasmic reticulum.     

Subcellular localization in normal and vitamin A-deficient rat liver of vitamin A serum transport proteins,albumin, ceruloplasmin and class I major histocompatibility antigens

The subcellular localization in rat liver cells of retinol-binding protein (RBP), prealbumin, ceruloplasmin, albumin, and class I transplantation antigen chains was investigated by radioimmunoassay determinations. The concentration of RBP was high in the rough and smooth endoplasmic reticulum (SER). The relative concentrations of prealbumin, ceruloplasmin and albumin were similar in the endoplasmic reticulum fractions and in the Golgi fraction. Neither of the proteins were found in significant amounts in the post-microsomal supernatant nor in the plasma membrane. The concentrations of the class I transplantation antigen chains were higher in the Golgi fraction than in the endoplasmic reticulum fractions. In the rough endoplasmic reticulum (RER) fraction ceruloplasmin and the class I antigens partially interact with high-molecular weight (MW) components, presumably membrane-bound glycosyltransferases. RBP, prealbumin and albumin seemed to be present in free form within the microsomal lumen. In vitamin A deficiency the RBP and to a lesser extent the prealbumin concentrations in the endoplasmic reticulum fractions were significantly increased, as compared to fractions from normal livers. This suggests that the presence of vitamin A is a prerequisite for the transport of RBP from the endoplasmic reticulum to the Golgi complex. The intracellular concentrations of albumin and ceruloplasmin were not significantly altered by vitamin A deficiency. In contrast, the amounts of the class I antigen heavy chains were found to be increased.     

Exchange of phospholipids between mitochondria and rough or smooth microsomes in vitro.

Phospholipids in mitochondria can be exchanged with those in two microsomal fractions from rough endoplasmic reticulum (rough microsomes) and smooth endoplasmic reticulum (smooth microsomes) in vitro in the presence of cell supernatant. The amounts of phospholipids transferred from each submicrosomal fraction to nitochondria were slightly different. The compositions of the phospholipids transferred to mitochondria from both microsomal fractions were the same, though these two fractions actually had different phospholipid compositions.     

STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN LIVERS OF PHENOBARBITAL-TREATED RATS : I. The Site of Activity of Acyltransferases Involved in Synthesis of the Membrane Phospholipid

The localization of acyltransferases involved in acylation of α-glycerophosphate, during phenobarbital induced proliferation of smooth endoplasmic reticulum (ser) membranes, has been investigated using cytochemical and cell fractionation techniques. In cytochemical studies of normal rat liver, reaction product marking acyltransferase activity was associated to the greatest extent with the rough endoplasmic reticulum (rer) membranes and to a lesser extent with ser membranes. In liver from phenobarbital-treated rats, reaction product was largely restricted to ser membranes. The specific activity of the acyltransferases of rough microsomes from normal rat liver was higher than that of the smooth microsomes. On injection of phenobarbital, this fell rapidly after three injections to a low level, at which it remained during subsequent treatment. The specific activity of the smooth microsomes, on injection of phenobarbital, rose to a peak 12 hr after the first injection, after which it fell to a level at an activity above that of smooth microsomes of normal liver. A mechanism is postulated for the biogenesis of smooth membranes in which the phospholipid is synthesized in situ and the protein is synthesized in the rer and moves to the site of newly synthesized phospholipid, where it is inserted to produce a whole membrane.     

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol- dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.